Antivirus proteins having a kringle 5 subunit

ABSTRACT

COVID-19 results from the infection of the SARS-CoV-2 virus and has spread quickly to literally infected the world. Although coronavirus spike proteins can recognize a broad range of host cell-surface proteins, inhibiting spike protein binding to a survival factor called GRP78 results in a significant reduction in SARS-CoV-2 attachment, entry and replication in lung and kidney cells. This inhibition is accomplished with a novel type of inhibitor that potently blocks the binding of SARS-CoV-2 spike protein and whole virus to surface-bound GRP78. These novel GRP78 inhibitors also down regulate cytokines (IL10, IL6), immune co-inhibitory checkpoint proteins (PD-L1, B7H3, B7H4), and up regulate immune co-stimulatory proteins (MHC-II, CD-86) resulting in the reduction of the immune suppressive nature of infected lung alveolar epithelial cells in vitro and in vivo. Finally, these novel GRP78 inhibitors inhibit the hyperfibrinolysis of infected lung cells by reducing the activation of plasmin on cell surfaces.

CLAIM OF PRIORITY AND INCORPORATION BY REFERENCE

This application claims priority from 63/012,900 filed Apr. 20, 2020. Tothe maximum extent permitted, as stated in application Ser. No.63/012,900, priority is claimed based on the family of then-copendingpatent applications, U.S. provisional patent application No. 62/584,564filed Nov. 10, 2017, and U.S. patent application Ser. No. 16/184,247filed Nov. 8, 2018, published as US 2019-0142913 A1 May 16, 2019. Theaforesaid publication and U.S. patent Ser. No. 10/905,750 areincorporated by reference as if fully set forth herein.

DESCRIPTION OF RELATED ART

COVID-19 is an abbreviation from “coronavirus disease 2019” that is aresult of an infection from a coronavirus called SARS-CoV-2. This viralinfection has been declared a pandemic by the WHO and is considered bythe CDC as a serious public health problem. Cases of COVID-19 areexploding worldwide and as of yet there is no approved cure. COVID-19can result in severe respiratory illness in patients with pre-existingconditions and in older adults leading to permanent lung damage anddeath.

Currently, the use of two anti-malaria drugs, chloroquine andhydroxychloroquine, have both shown promise in preventing SARS-CoV-2virus from infecting cells in the laboratory. Recently, in a smallnumber of preliminary clinical trials against COVID-19 usinghydroxychloroquine and an antibiotic showed that in the blood of treatedpatients the amount of virus was reduced much faster than in nontreatedpatients. These results are encouraging and even though the side-effectsof heart and nerve damage as well as suicidal thoughts are manageable,they are still disturbing. These studies have also not yet shown thatthe patients lived longer or were more likely to recover withhydroxychloroquine. Finally, in a very recent report another treatment,Leronlimab, which is an antibody against CCR5 has shown positive resultsin 8 patients. CCR5 inhibitors have been shown to reduce the COVID-19stimulated inflammatory cytokine storm in the lungs allowing for moretime for recovery. Leronlimab is not a cure or a vaccine. Again, thereis an urgent and unmet need for safe, effective new drugs to treatCOVID-19.

The off-label therapies described above are being used in the clinic forCOVID-19 patients but were not designed for specific inhibition ofSARS-CoV-2 virus attachment, entry and replication. Despite this, theseoff label therapies have shown some desirable responses. Although someresults look promising, many patients do not respond to these therapiesand many deaths worldwide are still happening. New drugs thatspecifically target the SARS-CoV-2 virus infection are badly needed. Ourwork to create novel GRP78 inhibitors for anti-cancer and anti-immunesuppression, could be one of the new targets and therapies for COVID-19that is badly needed.

Understanding the mechanism of how viruses and host cell-surfaceproteins interact could help define their tropism, pathogenicity andlead to potential new targets for inhibition. For example, recentpublications have described a role for surface-bound GRP78 during virusentry and replication. GRP78 was identified as a co-receptor forCoxsackievirus A9 and Dengue virus for attachment and entry. Inaddition, for the Japanese encephalitis virus (JEV), cell surface GRP78is important for viral entry and critical for virus replication.Surface-bound GRP78 is also known to serve as an attachment factor forfour betacoronaviruses, MERS-CoV, bCoV-HKU9, SARS-CoV, and SARS-CoV-2.FIG. 1 illustrates how coronaviruses use GRP78 as a co-receptor forattachment, internalization and replication. Virus infection of lungairway cells also results in the up regulation of GRP78 surfaceexpression, which leads to further attachment and enhanced viral entryof infected cells.

Although coronavirus spike proteins can recognize a broad range of hostcell-surface proteins, inhibiting GRP78, by either knockdown with siRNA,or cleavage with subtoxin A, or with an antibody, results in significantreduction in virus attachment, entry and replication. High expression ofGRP78 was shown to be on the surface of stressed epithelial andendothelial cells along the human airways. Recently, it has been shownthat cigarette smoke increased surface expression of GRP78 on stressedbronchial epithelial cells. Since COVID-19 has been shown to be worse inpeople that smoke, vape, have respiratory disease or are older, wesuspect the expression of surface GRP78 on lung epithelial cells issignificantly higher in this population. Even though expression of GRP78alone was not enough to render nonpermissive cells susceptible toMERS-CoV infection, it has been shown that GRP78 is critical for viralentry and replication.

BRIEF SUMMARY OF THE INVENTION

I have discovered that surface-bound GRP78 on A549 adenocarcinoma humanalveolar basal epithelial cells and VERO epithelial cells up regulatesimmune co-inhibitory checkpoint proteins, PD-L1, B7H3, B7H4 and downregulates immune co-stimulatory proteins, MHC-II and CD86. I have alsodiscovered that surface-bound GRP78 up regulates cytokines IL-10, IL6,on A549 adenocarcinoma human alveolar epithelial cells which results inthe blunting of the immune response. I have created a class of novel andpotent inhibitors that specifically bind to the N-terminal domain ofGRP78 that block the binding of SARS-CoV-2 virus to GRP78 and completelyreverses cytokine expression and the immune suppressive phenotype onlung epithelial A549 cells and VERO epithelial cells (FIG. 1B). Stressedepithelial cells up regulate surface bound GRP78 that not only acts as aco-receptor for SARS-COV-2 virus to assist in attachment, entry andreplication, but also blunts the immune response against SARS-COV-2virus and infected cells. The GRP78 inhibitor of the invention cansignificantly reduce the attachment, entry and replication of SARS-COV-2virus as well as reduce the immune suppressive nature of infected lungalveolar epithelial cells in vitro and in vivo.

DESCRIPTION

Dr. Dvorak published that a tumor is like a wound that won't heal. Avery general analogy is that the same kind of mechanism exists forSARS-CoV-2 infection where the infection in the lungs is like a woundthat won't heal. Viral infection induces enormous stress upon theinfected cells and increases expression of GRP78 as happens in the tumormicroenvironment (TME). The TME induces a pro-inflammatory, immunesuppressive tumor cells similar to what is observed with viralinfections on their target cells. In the co-pending GRP78 Antagonistapplication GRP78 inhibitors, Kr1Fc, K5Fc and K5 can block GRP78'sinteraction with cell surface receptors and decrease the immunesuppressive, inflammatory nature of tumor cells. We now show for thefirst time that our GRP78 inhibitors, Kr1Fc, K5Fc and K5 can block thebinding of SARS-CoV-2 spike protein to GRP78 with nM potency.Furthermore, our GRP78 inhibitors, Kr1Fc and K5, potently block wholelive virus, pseudotyped SARS-CoV-2, attachment and entry into VEROkidney epithelial cells. Specifically, the co-pending GRP78 Antagonistapplication teaches:

A surface-bound GRP78 inhibitor blocks SARS-COV-2 spike proteinattachment and entry. The co-pending GRP78 Antagonist applicationteaches inhibitors to GRP78 on endothelial and cancer cells. Theseinhibitors have now been tested against SARS-COV-2 virus binding toGRP78 and to human lung cells. A lead inhibitor, containing the kringledomain of ROR1 fused to a human IgG1 Fc domain (Kr1Fc), binds with highaffinity to the N-terminal domain of GRP78. The invention teaches thatKr1Fc, K5Fc and K5 potently block the binding of SARS-COV-2 spikeprotein to GRP78. The disclosed GRP78 inhibitors are effective atblocking the attachment, entry and replication of SARS-COV-2 pseudotypedvirus.

A new mechanism that promotes immune tolerance that is readilytargetable. Over expression of GRP78 in stressed cells leads to a largeincrease in surface-bound GRP78. The invention teaches thatsurface-bound GRP78 on human A549 adenocarcinoma alveolar basalepithelial cells induces the expression of A) cytokines IL-10, and IL-6,B) immune co-inhibitory checkpoint proteins, PD-L1, B7H3, B7H4, and C)suppresses the expression of immune co-stimulatory proteins, MHC-II, andCD86. By blocking GRP78 binding to surface proteins with Kr1Fc, theimmune-suppressive phenotype of A549 cells can be reversed. In theinvention, inhibition of GRP78 binding to SARS-COV-2 SPIKE PROTEIN willlead to a decrease in viral load, cytokine storm and immune suppressionassociated with SARS-COV-2 infection.

Novel GRP78 inhibitors that bind to the N-terminal domain of GRP78reduce surface GRP78 expression which is only expressed on stressedcells and not normal cells, leads to a safer therapy than othercurrently approved anti-viral therapies. The invention teaches potentinhibitors that bind tightly to the N-terminal domain of GRP78 resultingin the inhibition of SARS-CoV-2 virus binding. In the invention theinhibitors to surface-bound GRP78 are safe in CEREP receptor binding andin normal fibroblast proliferation assays. Previously practicedtherapies being used against COVID-19 like hydroxychloroquine, bindweakly to SARS-CoV-2's receptor ACE2 on lung epithelial cells. The factthat ACE2 is expressed on several other normal cells and thathydroxychloroquine has such a weak binding affinity, supports theoff-target side-effects of this drug seen in clinical trials. Anotherdrug approved for treatment of SARS-CoV-2 virus is Remdesivir.Remdesivir is an adenosine analogue that blocks mitochondrial RNApolymerase essential for virus replication. However, there are severaloff-target toxicities in the gut and lungs with Remdesivir and it toomust be used cautiously. This invention teaches GRP78, which is notexpressed on the surface of normal cells is a safer and more effectivetarget for therapies against COVID-19.

The invention also teaches that N-terminal GRP78 inhibitors blockSARS-CoV-2 virus induced hyperfibrinolysis and coagulopat+hy (“clotstorm”) through inhibition of plasmin generation (FIG. 2). Patients withsevere COVID-19 have comorbidities like hypertension, heart disease,diabetes, and cancer that are known to have serious coagulopathies.Hyperfibrinolysis, reflected by elevated serum D-dimer levels, waspresent in 97% of the severe COVID-19 patients before death. Plasmin, akey player in hyperfibrinolysis and as such D-dimer levels, alsoenhances the virulence and pathogenicity of the SARS-CoV-2 virus byclipping a furin site on its envelope protein. Kr1Fc, K5Fc, and K5 canalso block the activation of plasmin on lung epithelial and endothelialcell surfaces leading to reduced fibrinolysis, D-dimer formation andfurin site cleavage. Not only will our GRP78 inhibitors significantlyblock SARS-CoV-2 virus attachment, entry and replication but they alsoreduce the cytokine storm, inhibit hyperfibrinolysis and increase theimmune surveillance against SARS-CoV-2 virus (FIG. 2). The inventionteaches N-terminal binding GRP78 inhibitors either alone or incombination with other therapies will significantly reduce thepathogenicity of COVID-19.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of SARS-CoV-2 virus attachment, entry andreplication with and without GRP78 inhibitors.

FIG. 2 is a schematic of SARS-CoV-2 virus inhibition by our novel classof GRP78 inhibitors.

FIG. 3 is an analysis of Kr1Fc, K5Fc, Kr1 and K5 proteins purity,structure and binding to GRP78.

FIG. 4 A is a schematic that shows that N-terminal GRP78 inhibitorsblock binding of GRP78 to SARS-CoV-2 spike protein.

FIG. 4 B is a graph that show that N-terminal GRP78 inhibitors blockbinding of GRP78 to SARS-CoV-2 spike protein.

FIG. 5 A is a series of graphs that show that GRP78 inhibitors blockbinding of PE-labeled SARS-CoV-2 spike protein to A549 lung cells.

FIG. 5 B is two histograms that show that GRP78 inhibitors block bindingof PE-labeled SARS-CoV-2 spike protein to A549 lung cells.

FIG. 6 A is a series of graphs that show that GRP78 inhibitors preventSARS-CoV-2 spike protein-PE binding to VERO cells.

FIG. 6 B is two histograms that show that GRP78 inhibitors preventSARS-CoV-2 spike protein-PE binding to VERO cells.

FIG. 7 A is a series of graphs that show that Kr1Fc and K5 preventbinding of SARS-CoV-2 spike protein either with preincubation with VEROcells or added at the same time as the spike protein.

FIG. 7 B is two histograms that show that Kr1Fc and K5 prevent bindingof SARS-CoV-2 spike protein either with preincubation with VERO cells oradded at the same time as the spike protein.

FIG. 8 A is a series of graphs that demonstrate that N-terminal GRP78inhibitors reduce the expression of surface-bound GRP78 on VERO cells.

FIG. 8 B is two histograms of graphs that demonstrate that N-terminalGRP78 inhibitors reduce the expression of surface-bound GRP78 on VEROcells.

FIG. 9 A is a series of graphs that demonstrate that N-terminal GRP78inhibitors reduce the expression of surface-bound GRP78 on A549 lungcells.

FIG. 9 B is two histograms of graphs that demonstrate that N-terminalGRP78 inhibitors reduce the expression of surface-bound GRP78 on A549lung cells.

FIG. 10 A is a schematic that shows that surface-bound GRP78 expressionon VERO cells, which is significantly reduced by Kr1Fc and K5, isessential for SARS-CoV-2 spike protein internalization.

FIG. 10 B is a series of graphs that show that surface-bound GRP78expression on VERO cells, which is significantly reduced by Kr1Fc andK5, is essential for SARS-CoV-2 spike protein internalization.

FIG. 10 C is histograms that show that surface-bound GRP78 expression onVERO cells, which is significantly reduced by Kr1Fc and K5, is essentialfor SARS-CoV-2 spike protein internalization.

FIG. 11 A is a graph that demonstrates that GRP78 inhibition withN-terminal GRP78 binding proteins, Kr1Fc and K5, potently andsignificantly inhibit whole SARS-CoV-2 pseudotyped virus attachment andinternalization on VERO cells.

FIG. 11 B is a graph that demonstrates that GRP78 inhibition withN-terminal GRP78 binding proteins, Kr1Fc and K5, potently andsignificantly inhibit whole SARS-CoV-2 pseudotyped virus attachment andinternalization on VERO cells.

FIG. 12 A is a series of graphs which illustrate that K5 significantlyaugments co-inhibitory (PD-L1, B7H4) checkpoint protein andco-stimulatory (CD86, MHC-II) protein expressions induced by solubleGRP78 on A549 lung cells.

FIG. 12 B is a histogram which illustrates that K5 significantlyaugments co-inhibitory (PD-L1, B7H4) checkpoint protein andco-stimulatory (CD86, MHC-II) protein expressions induced by solubleGRP78 on A549 lung cells.

FIG. 13 A is a series of graphs that show that Kr1Fc significantlyaugments expression of co-inhibitory (PD-L1, B7H4) checkpoint proteinand co-stimulatory (CD86, MHC-II) protein expressions induced by solubleGRP78 on A549 lung cells.

FIG. 13 A is a histogram that shows that Kr1Fc significantly augmentsexpression of co-inhibitory (PD-L1, B7H4) checkpoint protein andco-stimulatory (CD86, MHC-II) protein expressions induced by solubleGRP78 on A549 lung cells.

FIGS. 14 A and B are histograms that demonstrate that Kr1Fc and K5inhibit soluble GRP78 induced cytokine expression of IL10 and IL6 fromA549 lung cells.

FIG. 15 A is a schematic that shows that Kr1Fc and K5 inhibit theactivation of human plasminogen on VERO cell surfaces.

FIG. 15 B is a histogram that shows that Kr1Fc and K5 inhibit theactivation of human plasminogen on VERO cell surfaces.

FIG. 16 is a graph that shows the mice treated with Kr1Fc (60 mg/kg) orK5 (90 mg/kg) every other day, intraperitoneally displayed no weightloses or overt toxicity.

FIG. 17 is a table showing cytotoxicity and antiviral activity forparticular sequences across GRP78 recognition sites.

DETAILED DESCRIPTION

My previous publications, incorporated by reference, teach that the 5thkringle domain of human plasminogen (K5) bound to surface GRP78 toinduce inhibition of tumor angiogenesis and tumor growth. However, K5was not considered to be a good drug candidate due to its unknownmechanism of action and poor half-life in mice and monkeys (<20 min). Bydetermining how soluble GRP78 binds to tumor cell surfaces, a novelGRP78 binding protein was identified and is called receptor tyrosinekinase-like receptor-1 (ROR1). Predictably, ROR1 has a kringle domainthat is very similar (>70%) to K5. The ROR1 kringle domain, Kr1, bindsto GRP78 100×s tighter (Kd=0.005 nM) than K5 (Kd=0.6 nM). The inventionalso shows that the ROR2 has a >70% homology to K5 and will have similaractivity as ROR1 kringle domain. As shown in FIG. 3, the inventiondiscloses novel kringle fusion protein that contains the ROR1 kringledomain, Kr1, and a human IgG1 Fc domain. The addition of an IgG Fcdomain to peptides has been shown to increase plasma half-life. We havenow expressed the fusion proteins, Kr1Fc and K5Fc in Expi293F HEK cellsand ExpiCHO cells and purified them over a Protein A column. The bindingof the recombinant Kr1Fc, Kr1, K5, and K5Fc proteins tobenzamidine-agarose as a second purification column suggests that thekringle domains are folded properly. At the time of this writing, I haveexpressed and purified about 600 mgs of Kr1Fc, 2 mgs of K5Fc, 10 mgs ofKr1, and 600 mgs of K5. The references incorporated by referencediscloses soluble GRP78 inhibitors Kr1Fc, K5Fc, Kr2Fc and Kr1 for use asanticancer agents.

Binding Inhibition Example #1

GRP78 Inhibitors Kr1Fc, K5Fc and K5 Inhibit SARS-CoV-2 Spike ProteinBinding to GRP78.

The prior art describes that GRP78 binds to the spike protein ofSARS-CoV-2 virus. The prior art does not disclose what the affinity ofbinding was for SARS-CoV-2 spike protein with GRP78. Using an ELISAassay, this invention examines that GRP78-HRP binds to plate boundSARS-CoV-2 spike protein with a Kd of 293+35 nM. To determine if Kr1Fc,K5Fc and K5 can block this binding, this invention teaches to coat a 96well plate with SARS-CoV-2 spike protein (100 nM) overnight at 4 C. Theplate was then blocked with skimmed milk at room temperature for 30 min.HRP-labeled GRP78 (200 nM) is then added to the plates with variousconcentrations of Kr1Fc, K5Fc, K5 and a negative control, unfoldedKr1Fc. The plates were then incubated at room temperature for 2 hours.Finally, the wells were washed with PBS and then 1-step ultra TMB-ELISAreagent was added to each well and incubated at room temperature for 60minutes and then read at 450 nM on a spectrophotometer. FIG. 4, showsthat Kr1Fc, K5Fc and K5 inhibit GRP78 binding to the SARS-CoV-2 spikeprotein with IC50 values of 0.35 nM, 28.5 nM and 460 nM respectively.The unfolded Kr1Fc negative control showed no inhibition of SARS-CoV-2spike protein binding to GRP78. This invention indicates that theinvention GRP78 N-terminal binding inhibitors potently block GRP78 andSARS-CoV-2 spike protein binding.

Binding Inhibition Example #2

GRP78 Inhibitors Kr1Fc and K5 Inhibit SARS-CoV-2 Spike Protein Bindingto A549 Alveolar Epithelial Adenocarcinoma Cells at 4 C.

In FIG. 5, this invention examines if Kr1Fc and K5 could block bindingof PE-labeled SARS-CoV-2 spike protein to A549 lung cells. A549 cells(50,000 cells/100 uL) were added to Eppendorf tubes in PBS. GRP78inhibitors, at various concentrations, and 50 nM PE-labeled SARS-CoV-2spike protein were added to the cells and incubated overnight at 4 C toblock internalization of the receptors. Cells were washed 2-times withPBS and run on a Guava PCA flow cytometer. The negative control used wasa human IgG1-PE antibody. Flow cytometry analysis shows thatSARS-CoV-2-PE spike protein bound to 52% of the A549 cells compared tocontrol IgG1-PE antibody at 23% of cells. Kr1Fc at 100 nM significantlyblocked the SARS-CoV-2-PE spike protein binding to A549 lung epithelialcells by greater than 99%, whereas K5 at 500 nM blocked theSARS-CoV-2-PE spike protein binding to A549 lung epithelial cells around70%. These results indicate that GRP78 is important for SARS-CoV-2 virusbinding to A549 lung epithelial cells.

Binding Inhibition Examples #3

GRP78 Inhibitors Kr1Fc and K5 Inhibit SARS-CoV-2 Spike Protein Bindingto VERO Cells at 37 C.

This invention examines if N-terminal GRP78 inhibitors could block thebinding of SARS-CoV-2 spike protein on VERO monkey epithelial kidneycells. VERO cells are known to have high expression of the SARS-CoV-2receptor, ACE2, and as such are very susceptible to SARS-CoV-2 virusinfection. This invention teaches that both pre-incubation of VERO cellswith GRP78 inhibitors and also by adding GRP78 inhibitors at the sametime as the SARS-CoV-2 spike protein results in significant and potentinhibition of spike protein binding. VERO cells (50,000/100 uL) in PBSwere added to Eppendorf tubes. Either Kr1Fc at 100 nM or K5 at 500 nMwere added to half of the tubes of VERO cells for a pre-incubation timeof 6 hrs. before 50 nM PE-labeled SARS-CoV-2 spike protein was added. Inthe other half of the tubes with cells, the PE-labeled SARS-CoV-2 spikeprotein was added at the same time as the GRP78 inhibitors listed above.The tubes of cells were incubated at 37 C with mild shaking. After 24hours, the cells were spun and washed twice with PBS. Fresh PBS wasadded to the cells and flow cytometry analysis of PE-labeled SARS-CoV-2spike protein bound to VERO cells was detected on a Guava PCA flowcytometer. In this invention GRP78 inhibitors Kr1Fc and K5, displayedinhibition of SARS-CoV-2 spike protein binding with both a 6-hourpre-incubation or no pre-incubation with GRP78 inhibitors (FIGS. 6 & 7).Kr1Fc at 100 nM displayed greater than 90% inhibition of SARS-CoV-2spike protein binding to VERO cells. K5 at 500 nM displayed an >80%inhibition of SARS-CoV-2 spike protein binding to VERO cells. Thepre-incubation of GRP78 inhibitors on VERO cells leads to increasedinhibition of SARS-CoV-2 binding.

Internalization of Surface-Bound GRP78 Example #4

Surface-Bound GRP78 is Significantly Decreased after Kr1Fc and K5Treatment.

In this invention, Kr1Fc (100 nM), and K5 (500 nM) were added separatelyto Eppendorf tubes containing either 50,000 A549 cells or 50,000 VEROcells in triplicate. The cells and GRP78 inhibitors were incubated at 37C for 24 hours. The cells were pelleted by centrifugation and washedtwice with PBS. Anti-GRP78 monoclonal antibody labeled with PE (1 mg/ml)was then added at 1 ul per tube. A negative control of a human IgG1-PEantibody was also added to a tube of untreated cells. After 1 hourincubation of the anti-GRP78-PE antibody at room temperature, cells werewashed twice with PBS and analyzed for surface-bound GRP78 by a Guavaflow cytometer. FIG. 8 demonstrates that the treatment of VERO cellswith GRP78 inhibitors, Kr1Fc and K5, significantly reduced theexpression of GRP78 on the surface of the cells. FIG. 9 shows thattreatment of A549 lung cells with GRP78 inhibitors, Kr1Fc and K5significantly reduced the concentration of surface-bound GRP78 bygreater than 80%. Comparing the expression of surface-bound GRP78 onA549 and VERO cells, it is clear the VERO cells have a much higher levelof surface-bound GRP78. Since these cells are more receptive for viralinfection, this invention and data show that it is not only the levelsof surface ACE2 but also GRP78 that is important for infectivity.Because these GRP78 inhibitors reduce the surface-bound GRP78 levels,does this result in the reduction of virus binding only or is GRP78 alsoresponsible for internalization of SARS-CoV-2 spike virus.

Internalization Inhibition Example #5

Kr1Fc and K5 Inhibit pHrodo-Red Labeled SARS-CoV-2 Spike ProteinInternalization in VERO Cells.

This invention examines if surface-bound GRP78 inhibition can inhibitbinding of SARS-CoV-2 spike protein and block its internalization. Asshown in FIG. 10A, SARS-CoV-2 spike protein was labeled with pHrodo-reddye, which has a weak fluorescence at pH 7 (cell surface), but has astrong fluorescence at pH 4 (inside cell) allowing for the detection ofspike protein internalization. VERO cells were pre-incubated with GRP78inhibitor, Kr1Fc and K5 for 6 hrs. After 6 hrs., Rodo-red labeledSARS-CoV-2 spike protein was added to the VERO cells and incubated at 37C with mild shaking for 24 hrs. Cells were then pelleted, washed twiceand flow cytometry analysis was performed on a Guava PCA flow cytometerto detect inhibition of internalized spike protein. In FIG. 10B, thisinvention shows that Rodo-red labeled SARS-CoV-2 spike protein isinternalized in about 52% of VERO cells in 48 hrs. at 37 C. When VEROcells are pre-incubated with the GRP78 inhibitor, Kr1Fc, or K5 theinternalization of Rodo-red labeled SARS-CoV-2 spike protein issignificantly inhibited by 90-95% similar to the Rodo-red labeled IgG1antibody negative control. In both examples, A549 and VERO cells GRP78inhibitors prevent SARS-CoV-2 spike protein internalization.

Whole SARS-CoV-2 Virus Neutralization Example #6

GRP78 Inhibitors, Kr1Fc and K5, Neutralize SARS-CoV-2 Pseudotyped VirusInfection of VERO Cells.

In this invention, modified SARS-CoV-2 virus assay was performed by IBTBioservices (Rockville, Md.). In this assay, a SARS-CoV-2 pseudotypedvirus was generated by replacing the replication RNA piece from theSARS-CoV-2 virus with RNA encoding the Luciferase enzyme protein fordetection. The remaining structural proteins (spike protein, envelopeprotein, and matrix protein and nucleocapsid protein) were left intact.This allows for SARS-CoV-2 (rVSV-SARS-CoV-2 (D614G)) pseudotyped virusto attach and internalize but not replicate. The invention teaches thatKr1Fc and K5 can prevent the full SARS-CoV-2 pseudotyped virus fromattaching and entry into VERO cells. In FIG. 11A, two compoundsdemonstrate potent activity with IC50 values of 3.5 uM for Kr1Fc, and47.4 uM for K5. The assay was performed by using eight dilutions from0.5 nM to 500 uM for K5 and 0.01 nM to 62 uM for Kr1Fc. These dilutionswere added to triplicate wells in a 96 well plate with VERO cells (1×10⁵cells per well). Wells in accordance with the invention are infectedwith SARS-CoV-2 pseudotyped virus at 25,000-35,000 Relative Light Unitsin each well. Plates were incubated for 24 hours, and attached cellswere washed and then each well was read for Luciferase activity usingBright-Glo Assay System Kit (Promega). The toxicity of the testcompounds was also determined in parallel against VERO cells withoutvirus (FIG. 10B). The 50% and 90% effective neutralization concentration(IC50, IC90) and 50% cell death concentration (cytotoxic, CC50) valuesare calculated by regression analysis to demonstrate efficacy. Theselectivity index (SI90) (CC90 divided by IC90), which is indicative ofthe safety window between cytotoxicity and antiviral activity for 1 loginhibition was calculated and presented in FIG. 17. The higher the SI90value, the more effective and safer the inhibitor.

Immune Suppression Inhibition Example #7

Kr1Fc, K5Fc and K5 reverses immune suppressive phenotype onadenocarcinoma alveolar lung epithelial cells (A549) induced by sGRP78binding. This invention addresses whether soluble and surface boundGRP78, which has been shown to be stimulated during viral infectionswould augment checkpoint protein expression on lung cells similar towhat has been reported with dendritic cells. To determine this, sGRP78(5 ug/ml) was added to A549 cells and grown for 3 days±Kr1Fc at 37 C/5%CO2. After 3 days, cells were fixed (not permeabilized), stained withfluorescently labeled antibodies and flow cytometry analysis wasperformed on co-inhibitor checkpoint proteins, PD-L1, B7H3, B7H4 andco-stimulatory proteins MHC-II, CD86. The present invention examines theexpression of surface GRP78 with and without Kr1Fc, K5Fc and K5inhibitors. We chose a concentration of 5 ug/ml sGRP78 because it hasbeen shown that sGRP78 circulates in cancer and Rheumatoid Arthritispatients around this concentration. FIGS. 12 and 13 show that sGRP78induced a significant increase in expression of immune inhibitorycheckpoint proteins PD-L1, B7H3, B7H4 and decreased expression of immunestimulatory proteins MHC-II and CD86 on A549 cells. Kr1Fc and K5completely reversed this immune-suppressive phenotype.

Cytokine Expression Inhibition Example #8

Previously, we have shown that soluble GRP78 induces cytokine expressionon tumor cells. In many ways, viral infection mimics the tumormicroenvironment, as such we determined that soluble GRP78 could upregulate expression of IL10 and IL6 on A549 lung cells. In FIG. 14, wenow show that Kr1Fc and K5 inhibit soluble GRP78 induced cytokineexpression of IL10 and IL6 from A549 lung cells. A549 cells (50,000)were added to Eppendorf tubes in full DMEM media. In half the tubes,soluble GRP78 (5 ug/ml) was added along with either K5 (500 nM) or Kr1Fc(100 nM). An IgG human antibody was used as a negative control. Afterincubation for 3 days at 37 C with mild shaking, the cells were spundown and the supernatant was tested using either an IL10 or IL6 ELISAassay kits (R&D Systems). The protocol for each kit was followed permanufacturer instructions and pg/ml of each cytokine at each conditionon A549 cells with and with GRP78 inhibitors was calculated fromstandard curves. A significant and dose dependent inhibition ofcytokines expression from A549 cells was observed with Kr1Fc and K5GRP78 inhibitors.

Plasminogen Activation Inhibition Example #9

Kr1Fc and K5 Block the Hyperfibrinolysis Induced by Plasmin Formation.

People with diabetes, hypertension, lung cancer and heart disease have ahigher risk of being infected by SARS-CoV-2 virus and a greater chanceof dying. The leading causes of death from COVID-19 is hemorrhage orbleeding disorders and that one of the characteristics of the disease isoveractivity of the system responsible for removing blood clots(hyperfibrinolysis). This aberrant coagulopathy is caused by elevatedlevels of plasminogen leading to plasmin. Recent publications show thatin all of the comorbidities that cause worse outcomes for people withCOVID-19, elevated levels of plasmin have been found to be a commonfactor. Plasminogen normally circulates in blood as an inactive protein.Once an injury or a lesion occurs (FIG. 2, FIG. 15A (red)), plasminogenis activated to its active form called plasmin. Plasmin then dissolvesblood clots at the lesions by clipping fibrinogen resulting infibrinogen fragments called D-dimers. More than 97% of peoplehospitalized with severe COVID-19 have increased levels of D-dimer.D-dimer levels are associated with the amount of virus detected in thebody and continue to rise as the severity of COVID-19 increases. InCOVID-19 survivors, D-dimer levels decreased to control levels.Higher-than-normal levels of plasmin and D-dimers can lead to severebleeding. Several publications have shown that plasminogen binds tosurface-bound GRP78 through one of its kringle domains, kringle 5 (K5).In fact, GRP78 binding to plasminogen increases its rate of activationto plasmin significantly. Since our GRP78 inhibitors, Kr1Fc, K5Fc andK5, consists of the plasminogen K5 kringle domain or a similar kringledomain (Kr1), we can show that these inhibitors significantly decreasethe activation of plasminogen to plasmin on lung cells by over 70% (FIG.15). This decrease in plasmin formation should result in reducedhyperfibrinolysis and D-dimer formation reducing the bleeding disordersobserved in severe COVID-19 patients.

Safety Profile Example #9

Kr1Fc, and K5 Show No Adverse Binding to Receptors and Ion ChannelProteins or Toxicity on Primary Human Cells.

To identify possible toxicities, a receptor binding profile assay with75 receptor and ion channels, and a cytotoxicity assay with 5 humanprimary cell lines (validated from single donor sources) with Kr1Fc (10uM) or K5 (100 uM) were determined by CEREP/Eurofins. No specificbinding or toxicity was observed, indicating that Kr1Fc exhibits a safe,selective biochemical profile and is unlikely to have adverse effects invivo.

Safety Profile Example #10

No Weight Loses or Overt Toxicity was Observed in Mice Treated GRP78Inhibitors Kr1Fc and K5 at 60 mg/kg and 90 mg/kg Respectively.

This invention examines the toxicity of GRP78 inhibitors Kr1Fc and K5 inBALB/c mice 8 weeks old. Three groups of mice were dosed intraperitoneal(i.p.) in a volume of 10 mL/kg scaled to the body weight of eachindividual animal. Treatment groups were as follows:

Group 1 received vehicle (PBS pH 7.2).

Group 2 received Kr1Fc at 60 mg/kg, i.p., every other day (qod) untilday 26.

Group 3 received K5 at 90 mg/kg every other day (qod) until day 26.

Animals were weighed daily on Days 1-5, and then twice weekly until day26. The mice were observed frequently for overt signs of any adverse,treatment-related (TR) side effects, and clinical signs were recordedwhen observed. Individual body weight was monitored as per protocol, andany animal with weight loss exceeding 30% for one measurement orexceeding 25% for three consecutive measurements was euthanized as a TRdeath. Group mean body weight loss was also monitored according toCharles River Discovery Services protocol. Acceptable toxicity wasdefined as a group mean body weight (BW) loss of less than 20% duringthe study and no more than 10% TR deaths. Deaths were classified as TRif it was attributable to treatment side effects as evidenced byclinical signs and/or necropsy.

FIG. 16 shows that Kr1Fc and K5 showed no overt toxicity or weight lossin mice dosed as listed above. These results support the CEREP/Eurofinsin vitro toxicity testing from above and suggest that the inventionlisted types of GRP78 inhibitors will be safe, with low toxicity inclinical studies.

Chemistry, Manufacturing, and Controls (CMC) Aspects for the Developmentof K5Fc, K5 and Kr1Fc.

According to the invention the practitioner will express and purifyKr1Fc, K5Fc, K5 and perform CMC assays to validate the purity, potency,and efficacy of Kr1Fc, K5Fc and K5 lots for in vitro and in vivoSARS-CoV-2 virus inhibition studies. Currently, Kr1Fc, K5Fc and K5 aretransiently expressed in Expi293 and ExpiCHO cells. Mammalian cellexpression is necessary due to the need for a correctly folded kringledomains and glycosylation of the Fc domain. In accordance with theinvention stable CHO clones expressing Kr1Fc, K5Fc and K5 will becreated and banked, for regulatory filing. In accordance with theinvention a practitioner will validate assays for purity, potency andidentity of lots of Kr1Fc, K5Fc and K5. To validate Kr1Fc's, K5Fc's andK5's lot-to-lot quality, a negative control of denatured Kr1Fc and apositive standard lot of Kr1Fc are banked and used in each assay forcomparisons.

Statistical Analysis:

In accordance with the invention assays in Chemistry, Manufacturing, andControls (CMC) aspects are run in triplicate on two separate occasionsby two different investigators. The average mean value for absorbance,density and EC50 values plus the standard deviation are calculated forall assays. Any compound lot that is significantly (p<0.05, by student'stwo-tailed t-test assuming normal distribution and equal variance)different than our control standard lot will not be used for furthertesting.

Lot to Lot Purity, Potency and Variability:

According to the invention the purity for Kr1Fc, K5Fc and K5 lots aredetermined by practicing 280 nm OD measurements for drug concentration,densitometry analysis (GelQuant) of Coomassie-stained SDSPAGE gels fordrug purity and mass spectrometry analysis (fee for service) for drugquality. These assays are used to ensure the desired quality of Kr1Fc ismaintained. For all in vitro studies, negative controls are Kr1Fc mixedwith denatured, dead Kr1Fc at various ratios (10% to 90%) to define thelimits of each assay and for a positive control, a master lot of Kr1Fcis set aside for comparison in all assays between different lots.

According to the invention the potency of Kr1Fc, K5Fc and K5 lots aredetermined using a direct binding ELISA assay between Kr1Fc, or K5Fc, orK5 and GRP78. Full-length his-tagged GRP78 (StressMart) at 100 nM in PBSis attached to nickel-coated 96 well plates (Pierce). Variousconcentrations of inhibitors between 0.5 to 50 nM are bound at 4 C.Plates are blocked with 10% BSA and washed with PBS containing 0.05%Tween®-20 detergent per manufacturer's instructions (Pierce). Finally, amouse anti-human Fc HRP-labeled antibody is added, and detection ofbound Kr1Fc, K5Fc or K5 is assessed with the TMB ELISA (Sigma) reagent.A secondary binding assay for Kr1Fc, K5Fc, and K5 lots are performedmeasuring Kr1Fc competition binding between SARS-COV-2 spike proteinsand GRP78, as was described in our results (FIG. 4).

According to the invention functional efficacy of Kr1Fc lots isdetermined by examining immune co-inhibitory checkpoint (PD-L1, B7H3,B7H4) and immune co-stimulatory (CD86, MHC-II) protein expression. Asdescribed in FIG. 5, sGRP78 (5 ug/ml) are added to lung epithelialadenocarcinoma A549 cells and grown for 3 days+Kr1Fc at 37 C/5% CO2.After 3 days, cells are removed by Versene, fixed, and then stained withfluorescently labeled antibodies and flow cytometry analysis areperformed.

Assays that have a signal-to-noise ratio greater than two-fold and areable to detect >10% impurities, and assays with <200 nM EC(50) valuesfor potency and functional assays are considered valid.

With respect to drug purity and identity, mass spectrometry can detectimpurities below 5% reliably. The two-fold signal-to-noise requirementhas been met for all assays except the functional checkpoint proteinexpression assays. An alternate approach is to test for proteinphosphorylation in signaling pathways with A549 cells using Kr1Fc.Protein phosphorylation assays with signaling pathways through PI3K,SMAD2/3, STAT3 are known in the prior art. These signaling pathwayassays lead to a rapid and accurate method to test for activity andfunctionality in lots of Kr1Fc.

According to the invention efficacy of GRP78 inhibitors, Kr1Fc, K5Fc,and K5, for blocking SARS-CoV-2 spike protein binding andinternalization effects on lung epithelial A549 and VERO cells has beendetermined and validated.

The disclosed Kr1Fc, K5Fc and K5 inhibitors potently block binding ofthe SARS-CoV-2 spike protein to GPR78 (FIG. 4A). Much like tumors, viralinfections create an inflammatory, immune evasive microenvironment. Thereferences incorporated by reference shows that stressed cells relocateGRP78 to cell surfaces. Surface-bound GRP78 induces immune suppressionby up regulating expression of co-inhibitory checkpoint proteins anddown regulating expression of co-stimulatory proteins. GRP78 alsoincreases the expression of inflammatory cytokines like IL-10, and IL-6.By blocking GRP78's activity and binding on lung, kidney epithelial andendothelial cell surfaces with our GRP78 inhibitors, one practicing theinvention can completely reverse the immune suppressive, inflammatorynature of human alveolar lung epithelial adenocarcinoma A549 cells, VEROepithelial and endothelial cells. In addition, studies show that theblocking of the N-terminal domain of GRP78 reduces the attachment, entryand replication of several types of viruses including betacoronaviruses.As such, we show in these studies that our GRP78 inhibitors, which bindto the N-terminal domain of GRP78, will produce similar effects as siRNAor an N-terminal GRP78 antibody against the SARS-CoV-2 virus.

According to the invention neutralization activity against fullpseudotyped SARS-CoV-2 virus (rVSV-SARS-CoV-2 (D614G)) of Kr1Fc and K5are able to prevent the virus from attaching and entry into VERO cellsgrown in culture. Two compounds demonstrate potent activity with IC(50)values of 3.448 uM for Kr1Fc, and 47.35 for K5. Eight dilutions from 0.5nM to 500 uM for K5 and 0.01 nM to 62 uM for Kr1Fc were added totriplicate wells with cells. Wells in accordance with the invention areinfected with SARS-CoV2 pseudotyped virus at 25,000-35,000 RelativeLight Units to each well. Plates were incubated for 24 hours, attachedcells were washed and then each well was read for Luciferase activityusing Bright-Glo Assay System Kit (Promega). The toxicity of the testcompound was also determined in parallel against VERO cells withoutvirus. The 50% and 90% effective neutralization concentration (IC50,IC90) and 50% cell death concentration (cytotoxic, CC50) values arecalculated by regression analysis to demonstrate efficacy. Theselectivity index (SI50) (CC50 divided by IC50), which is indicative ofthe safety window between cytotoxicity and antiviral activity wascalculated and presented in Table 1. The higher the SI50 value, the moreeffective and safer the inhibitor.

In accordance with the invention potent inhibition of virus attachmentis demonstrable. GRP78 is surface-bound in stressed and tumor cells, inaccordance with the invention the practitioner may add GRP78 to themedia of A549 and VERO cells to produce a cell line for virus attachmentand internalization. In accordance with the invention K5, K5Fc and Kr1Fcwill reduce surface-bound GRP78 by >90%. In accordance with theinvention, the reversal of immune suppression, observed with A549 cellstreated Kr1Fc, demonstrates a response which could reduce viralpathology in vivo.

In accordance with the invention Kr1Fc will inhibit an adapted SARScoronavirus in an acute respiratory distress syndrome lethal mouse model(BALB/c mice). SARS-COV-2 virus creates a severe acute respiratorysyndrome disease that is highly lethal. To date there have been no drugsdirectly approved for curing betacoronavirus infections. Part of thereason for this is due to the lack of appropriate animal models for drugtesting. Researchers are testing multiple animal models in macaques,marmosets, hamsters, cats, and ferrets with SARS viruses but few mimicsthe SARS-CoV-2 virus pathogenicity. Recently, Day et al. have adaptedand characterized a new strain of SARS-CoV (v2163) that targets lungsand is highly lethal in BALB/c mice. This model largely mimics the humanCOVID-19 disease. Because of the low expense, ease of handling andminimal amount of drug required, this model when practiced in accordancewith the invention demonstrates GRP78 inhibitor with a human SARS-CoVvirus. Since mouse and human GRP78 are 98% identical, studies of thisnature demonstrate Kr1Fc drug will block GRP78's activity in mice aswell as humans resulting in a greatly reduced infection of lung cells.

Experiment

In accordance with the invention the anti-viral activity of Kr1Fc and K5is demonstrated using a mouse adapted strain of the Urbani SARS-CoVcalled V2163. Half male and half female BALB/c mice are inoculated with50 uL containing 10⁴ CCID50 (Cell culture infectious dose 50% endpoint)of SARS-CoV-V2163 virus by intranasal (i.n.) delivery. Four groups ofmice (10 mice per group) are given 100 mg/kg/day K5, 50 mg/kg/day Kr1Fc,and PBS, pH 7.4 negative control, and a positive control for this modelused meeting the standards of the Institution for Antiviral Research.All Kr1Fc and K5 samples are dosed i.p., QD between shoulder blades 16hours prior to i.n. infection for the next 7 days. Mice are observeddaily, and group weights are taken throughout the study. Mice areobserved for death up to day 21 post virus exposure. Animals that losegreater than 30% of their initial body weight are humanely euthanizedand the day of euthanasia designated as the day of death. Lungs fromsacrificed mice are observed for gross pathology and discoloration andassigned a score ranging from 0 (normal appearing lung) to 4 (maximalplum coloration in 100% of lung). Mouse lung samples from each testgroup are pooled and homogenized in MEM solution and assayed for A)infectious virus using the virus yield assay, B) cytokine analysis(IL-6, IL-1 and IL-10) and immune cell infiltrates (NK, Macrophage,T-cell and DCs). Virus titer, cytokine concentration and immune cellnumbers are compared to controls by analysis of variance on logtransformed values assuming equal variance and normal distribution. Asignificant (p<0.05) improvement in survival with Kr1Fc or K5 treatmentcompared to PBS negative control and a significant decrease inviral-induced CPE in the lungs of provides a foundation for furtherstudies.

As described in the references incorporated by reference K5 has ahalf-life around 20 min and an MTD greater than 660 mg/kg in mice andmonkeys. The more potent GRP78 inhibitors, Kr1Fc, and K5Fc in accordancewith the invention and publication of proteins with an added Fc domainhave improved half-lives and may be advantageous over the individualkringle domains. In addition, combination therapy with an ACE2 inhibitorand our GRP78 inhibitor could demonstrate synergistic effects.

In accordance with the invention, the conclusions described aredemonstrated by statistical analysis. More specifically, graphgeneration and statistical analysis are done on GraphPad Prism 7.0software. Statistical comparisons are performed two-way ANOVA withBonferroni posttest by Student's two tailed t-test, assuming normaldistribution and equal variance, where differences are consideredsignificant at p<0.05. Power analysis with a Wilcoxon-Mann-Whitney testfor a two-sided unpaired sample power analysis using the G*Power 3.1.9.4program is used to determine the number of mice needed. The group size(n=10) is powered to detect decreases of at least 30% in the number ofmetastases between control and 10 mg/kg Kr1Fc treated-groups, assuming acoefficient of variation equal to 1.5 (as suggested byprojected/anticipated data), and using a two-sample t-test for lognormal data with 80% power and a significance level of 0.05. Maximum dayof death (MDD), cytokine values, and gross lung scores are analyzed byMann-Whitney pairwise comparisons or the Kruskal-Wallis test followed byDunn's multiple comparison test as applicable. Raw survival numbers arecompared by the Fisher exact test. Survivor curve analysis are doneusing the Kaplan-Meier method and a log rank test. When that analysisrevealed significant differences among the treatment groups, pairwisecomparisons of survivor curves are analyzed by theGehan-Breslow-Wilcoxon test, and the relative significance adjusted to aBonferroni-corrected significance threshold for the number of treatmentcomparisons made. Differences in percent weight loss are tested byone-way ANOVA with Newman-Keuls multiple comparison test, assuming equalvariance and normal distribution. For lung titer data, we will perform aKS test for normality, then use non-parametric Kruskal-Wallis test withDunn's multiple comparison test for groups that are not normallydistributed, and a one-way ANOVA with Newman-Keuls multiple comparisontest for groups that are normally distributed.

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1 are schematics of SARS-CoV-2 virus attachment, entry andreplication with and without GRP78 inhibitors. FIG. 1A shows stressinduces GRP78 to translocate to the cell surface. 1) SARS-CoV-2 virusattaches to GRP78 and ACE2. 2) GRP78 enhances SARS-CoV-2 virus entryinto the lung epithelial cells. 3) GRP78 increases replication andsecretion of SARS-CoV-2. FIG. 1 B shows GRP78 inhibitors blocks asignificant amount of SARS-CoV-2 virus binding and reducessurface-expression of GRP78, which then leads to a reduced amount ofSARS-CoV-2 virus entry into the cells. Finally, the lack of GRP78 oncell surfaces inhibits SARS-CoV-2 virus attachment, internalization andreplication. The red X indicates that blocking GRP78 binding on the cellsurface blocks viral replication

FIG. 2 is a schematic of SARS-CoV-2 virus inhibition by our class ofGRP78 inhibitors. This invention shows that N-terminal GRP78 inhibitorsblock 1) SARS-CoV-2 virus attachment and entry, 2) reduceshyperfibrinolysis by blocking the conversion of plasminogen to plasminon lung cell surfaces, 3) reverses immune suppression by reducingcheckpoint protein expression on lung and endothelial cells and 4)reduces the virus induced cytokine storm by reducing IL10 and IL6cytokine expression.

FIG. 3 is an analysis of Kr1Fc, K5Fc, Kr1 and K5 Proteins Purity,Structure and Binding. FIG. 3 A-D are image of SDS-PAGE analysis showingA) Kr1Fc, B) K5Fc, C) Kr1 and D) K5 proteins expressed in 293F HEKcells. Kringle proteins were purified by either protein A-agarose orbenzamidine-agarose. Lane markers are Mw: Protein marker, Lane 1:Reducing conditions, Lane 2: Nonreducing conditions, M: expressionmedia, F: flow through, W: wash, P: purified protein. A FIG. 3 E is agraphic depiction of Kr1-Fc and K5Fc fusion proteins with 2 kringledomains fused to a human IgG1 Fc domain. A FIG. 3 F is a schematicillustration of human GRP78 regions where published GRP78 proteininhibitors and the invention proteins interact. Known and predictedbinding sites of viruses to GRP78 protein are shown underneath the GRP78schematic. (ATPase domain SBD: substrate binding domain, KDEL; 4 residueC-terminal peptide).

FIG. 4 shows that N-terminal GRP78 inhibitors block binding of GRP78 toSARS-CoV-2 spike protein. FIG. 4 A is a Schematic of a binding assaywith full-length SARS-CoV-2 spike protein, HRP-labeled GRP78 and GRP78inhibitors in a 96 well plate. FIG. 4 B is a graph showing data thatN-terminal GRP78 inhibitors, Kr1Fc, K5Fc, and K5 can potently blockGRP78 binding to SARS-CoV-2 spike protein. Full length SARS-CoV-2 spikeprotein was bound to a 96 well plate overnight at 4 C. After blockingand washing, HPR-labeled GRP78 and various concentrations of Kr1Fc,K5Fc, K5 and a negative control, unfolded Kr1Fc were added to plates andincubated for 2 hrs. Wells were washed and TMB reagent was added andabsorbance measured by manufactures protocol. Each point is an averageof triplicate wells.

FIG. 5 shows that GRP78 inhibitors block binding of PE-labeledSARS-CoV-2 spike protein to A549 lung cells. SARS-CoV-2 spike protein-PE(50 nM) was added to 50,000 A549 lung cells with and without Kr1Fc (100nM) or K5 (500 nM). Cells were incubated overnight at 4 C to blockinternalization of the SARS-CoV-2 spike protein-PE. Cells were washed 2×with PBS and run on a Guava PCA flow cytometer. Control was a humanIgG1-PE Ab. FIG. 5 A shows Raw flow cytometry plots and FIG. 5 B is ahistogram overlay analysis of A549 cells with bound SARS-CoV-2 spikeprotein-PE competed with GRP78 inhibitors demonstrate that Kr1Fc and K5block SARS-CoV-2 spike protein-PE binding to A549 lung cells by over 99%and 65% respectively.

FIG. 6 shows that GRP78 inhibitors prevent SARS-CoV-2 spike protein-PEbinding to VERO cells. VERO cells were used due to their high expressionof ACE2 and their ability to be very susceptible to SARS-CoV-2 virusinfectivity. SARS-CoV-2 spike protein-PE (50 nM) was added to 50,000VERO kidney cells with and without Kr1Fc (100 nM) or K5 (500 nM). Cellswere incubated overnight at 37 C. Cells were then washed 2× with PBS andrun on a Guava PCA flow cytometer. Control was a human IgG1-PE Ab. FIG.6 A is dot blot flow cytometry analysis and FIG. 6B is a histogramoverlay of VERO cells that bound SARS-CoV-2 spike protein-PE with andwithout Kr1Fc and K5. From the data it is clear that Kr1Fc (100 nM) andK5 (500 nM) significantly reduced the binding of SARS-CoV-2 spikeprotein-PE to the surface of VERO cells by about 50%.

FIG. 7 shows that Kr1Fc and K5 prevent binding of SARS-CoV-2 spikeprotein either with preincubation with VERO cells or added at the sametime as the spike protein (FIG. 6). FIG. 7A shows dot blot flowcytometry analysis and histogram overlay analysis of SARS-CoV-2 spikeprotein-PE (50 nM) binding to VERO cells preincubated with or withoutKr1Fc (100 nM) or K5 (500 nM) for 6 hours. The inhibition of SARS-CoV-2spike protein-PE binding was greater than 60% with Kr1 or K5preincubation. These data suggest that preincubation of cells with GRP78inhibitors may reduce spike protein binding slightly better.

FIG. 8 demonstrates that N-terminal GRP78 inhibitors reduce theexpression of surface-bound GRP78 on VERO cells. To help define if ourGRP78 inhibitors simply block the binding to SARS-CoV-2 spike protein toGRP78 or actually remove GRP78 from the cell surface, resulting in lesssurface-bound GRP78 for spike protein binding and internalization, wemeasured the amount of surface-bound GRP78 on VERO cells after Kr1Fc(100 nM) and K5 (500 nM) treatment. FIG. 8 A shows Flow cytometryanalysis of VERO cells surface-bound GRP78 using a C-terminal GRP78antibody-PE. This antibody has not been shown to inhibit SARS-CoV-2spike protein binding. FIG. 8B shows the flow cytometry dot blot andhistogram overlay analyses Kr1Fc and K5 produce a greater than 50%decrease in surface-bound GRP78. This suggests that these N-terminalGRP78 inhibitors may internalize/remove GRP78 from VERO cell surfacesresulting in less binding sites for SARS-CoV-2 spike protein binding.

FIG. 9 demonstrates that N-terminal GRP78 inhibitors reduce theexpression of surface-bound GRP78 on A549 lung cells. To help define ifour GRP78 inhibitors can internalize/remove GRP78 from A549 lung cellsurfaces like VERO cells, we measured the amount of surface-bound GRP78on A549 cells after Kr1Fc (100 nM) and K5 (500 nM) treatment. FIG. 9 Ashows flow cytometry analysis of A549 cells surface-bound GRP78 shows amuch lower expression of surface-bound GRP78 than VERO cells. Again,suggesting why VERO cells may be much more susceptible to infection thanA549 cells. FIG. 9 B shows the flow cytometry dot blot and histogramoverlay analyses, Kr1Fc and K5 produce a decrease in surface-bound GRP78from 84% to 60% respectfully. This suggests that these N-terminal GRP78inhibitors may internalize/remove GRP78 from A540 and VERO cell surfacesresulting in less binding sites for SARS-CoV-2 spike protein binding.

FIG. 10 shows that surface-bound GRP78 expression on VERO cells, whichis significantly reduced by Kr1Fc and K5, is essential for SARS-CoV-2spike protein internalization. In FIG. 10 A, an assay, a pHrodo red dyewas used to label SARS-CoV-2 spike protein to measure internalization.pHrodo dyes have a weak fluorescence at pH 7 (outside or bound to cells)but have a strong fluorescence inside the cell at a pH around 5.5 to4.5. This allows for the measurement of internalization of the pHrodored-labeled SARS-CoV-2 spike protein on VERO cells. FIG. 10 B shows flowcytometry analysis of SARS-CoV-2 spike protein-pHrodo internalization onVERO cells with and without Kr1Fc and K5. From the dot blot analysis andthe FIG. 10 C histogram overlay plots, Kr1Fc (100 nM) and K5 (500 nM)significantly reduce the internalization of SARS-CoV-2 spikeprotein-pHrodo by over 95% in VERO cells.

FIG. 11 demonstrates that GRP78 inhibition with N-terminal GRP78 bindingproteins, Kr1Fc and K5, potently and significantly inhibit wholeSARS-CoV-2 pseudotyped virus attachment and internalization on VEROcells. In this invention, modified SARS-CoV-2 virus assay was performedby IBT Bioservices (Rockville, Md.). In this assay, a SARS-CoV-2pseudotyped virus was generated by replacing the replication RNA piecefrom the SARS-CoV-2 virus with RNA encoding the Luciferase enzymeprotein for detection. The remaining structural proteins (spike protein,envelope protein, and matrix protein and nucleocapsid protein) were leftintact. This allows for SARS-CoV-2 (rVSV-SARS-CoV-2 (D614G)) pseudotypedvirus to attach and internalize but not replicate. The invention teachesthat Kr1Fc and K5 can inhibit the full SARS-CoV-2 pseudotyped virus fromattaching and entry into VERO cells. FIG. 11 A shows potent, dosedependent and complete neutralization (99.9%) of SARS-CoV-2 pseudotypedvirus infectivity with IC₅₀ values of 3.5 uM for Kr1Fc, and 47.4 uM forK5 was observed. The assay was performed by using eight dilutions from0.5 nM to 500 uM for K5 and 0.01 nM to 62 uM for Kr1Fc. These dilutionswere added to triplicate wells in a 96 well plate with VERO cells (1×10⁵cells per well). Wells in accordance with the invention are infectedwith SARS-CoV-2 pseudotyped virus at 25,000-35,000 Relative Light Unitsin each well. Plates were incubated for 24 hours, and attached cellswere washed and then each well was read for Luciferase activity usingBright-Glo Assay System Kit (Promega). As shown in FIG. 11 B thetoxicity of the test compounds was also determined in parallel againstVERO cells without virus. Kr1Fc displayed no cellular toxicity up to 2mM and K5 inhibited VERO cells growth at a cytotoxic cell concentrationat 50% (CC50) of 375 uM. These toxicity numbers are at least 10-foldhigher than the virus inhibition concentrations indicating toxicity isnot the mechanism of action for virus attachment and entry inhibition.

FIGS. 12 A and B illustrate K5 significantly augments co-inhibitory(PD-L1, B7H4) checkpoint protein and co-stimulatory (CD86, MHC-II)protein expressions induced by soluble GRP78 on A549 lung cells. Flowcytometry histogram plots of checkpoint protein expression +/−sGRP78 (5ug/m) and K5 (500 nM). A549 cells were incubated for 3 days+/−sGRP78 (5ug/ml) and K5 (500 nM). The cells were then fixed and stained withfluorescently labeled antibodies as indicated and FACs analysis wasperformed on a Guava PCA. All antibodies were mouse anti-human and PElabeled. A mouse IgG-PE antibody (grey) was used as a negative control.Average of 3 independent analyses with different A549 cell split numbersbetween 5-10 were used. ns: not significant, * p<0.05, ** p<0.01,***p<0.005, ****p<0.001. Unless otherwise explained in context,asterisks “*” correspond to those in the figures.

FIGS. 13 A and B show that Kr1Fc significantly augments expression ofco-inhibitory (PD-L1, B7H4) checkpoint protein and co-stimulatory (CD86,MHC-II) protein expressions induced by soluble GRP78 on A549 lung cells.Flow cytometry histogram plots of checkpoint protein expression+/−sGRP78 (5 ug/m) and Kr1Fc (100 nM). A549 cells were incubated for 3days+/−sGRP78 (5 ug/ml) and Kr1Fc (100 nM). The cells were then fixedand stained with fluorescently labeled antibodies as indicated and FACsanalysis was performed on a Guava PCA. All antibodies were mouseanti-human and PE labeled. A mouse IgG-PE antibody (grey) was used as anegative control. Average of 3 independent analyses with different A549cell split numbers between 5-10 were used. ns: not significant, *p<0.05, ** p<0.01, ***p<0.005, ****p<0.001

FIGS. 14 A and B demonstrate that Kr1Fc and K5 inhibit soluble GRP78induced cytokine expression of IL10 and IL6 from A549 lung cells. A549cells (50,000) were added to Eppendorf tubes in full DMEM media. In halfthe tubes, soluble GRP78 (5 ug/ml) was added along with either K5 (500nM) or Kr1Fc (100 nM). An IgG human antibody was used as a negativecontrol. After incubation for 3 days at 37 C with mild shaking, thecells were spun down and the supernatant was tested using either an IL10or IL6 ELISA assay kits (R&D Systems). The protocol for each kit wasfollowed and pg/ml of each cytokine for each condition for A549 cellswith and with GRP78 inhibitors was calculated from standard curves. Eachline is an average of 3 wells. ****p<0.001

FIG. 15 shows that Kr1Fc and K5 inhibit the activation of humanplasminogen on VERO cell surfaces. In FIG. 15 A the schematic ofundamaged lung on left side with effective and smooth blood flow. Onright side is the damaged lung from SARS-CoV-2 virus infection. Theinfection causes intense inflammation, and hyperfibrinolysis inducingmicrothrombi and high levels of D-dimer. The red Plasminogen to plasminreaction is where the GRP78 inhibitors block the formation ofmicrothrombi and D-dimer by inhibiting cell surface plasmin activation.FIG. 15 B shows significant inhibition of plasmin activation by Kr1 andK5 on VERO cells. In a 96 well plate, VERO cells were added (50,000 perwell) and incubated overnight at 37 C, 5% CO2. The next day the mediawas removed and PBS was added to each well. GRP78 inhibitors, Kr1Fc at(100 nM, 10 nM) and K5 at (500 nM, 50 nM) plus human plasminogen (100ng) was then added to each well. Finally, plasmin substrateVAL-Leu-Lys-pNA (S2251) was added to each well per manufacturesinstructions and the plate was read at 405 nm to measure pNA formationwhich is proportional to the enzymatic activity of plasmin. As shown inthe chart, Kr1Fc and K5 significantly inhibited the Vmax for plasminogenactivation on VERO cells.

FIG. 16 shows the mice treated with Kr1Fc (60 mg/kg) or K5 (90 mg/kg)every other day, intraperitoneally displayed no weight loses or overttoxicity. Kr1Fc and K5 were dosed in BALB/c mice 8 weeks old. Threegroups of mice were dosed intraperitoneal (i.p.) in a volume of 10 mL/kgscaled to the body weight of each individual animal. Treatment groupswere; 1 group treated PBS, i.p. every other day until day 22; 1 grouptreated with Kr1Fc at 60 mg/kg, i.p., every other day (qod) until day22; and 1 group received K5 at 90 mg/kg every other day (qod) until day22. Animals were weighed daily on Days 1-5, and then twice weekly untilday 22. The mice were observed frequently for overt signs of anyadverse, treatment-related (TR) side effects, and clinical signs wererecorded when observed. Individual body weight was monitored as perprotocol, and any animal with weight loss exceeding 30% for onemeasurement or exceeding 25% for three consecutive measurements waseuthanized as a TR death. Group mean body weight loss was also monitoredaccording to Charles River Discovery Services protocol. Acceptabletoxicity was defined as a group mean body weight (BW) loss of less than20% during the study and no more than 10% TR deaths. Deaths wereclassified as TR if it was attributable to treatment side effects asevidenced by clinical signs and/or necropsy. From FIG. 16, it is clearthat Kr1Fc and K5 showed no overt toxicity or weight loss in mice dosedas listed above.

FIG. 17 is a table showing cytotoxicity and antiviral activity forparticular sequences across GRP78 recognition sites. As more fullydescribed below, sequence alignment of viruses is compared to knownGRP78 binding kringle sequences to identify possible GRP78 binding sitesto enable predicting which viruses will be responsive to our method oftreatment for viral inhibition through blocking the N-terminal domain ofGRP78.

Treatment Parameters when Using the Therapeutic Invention

Severe viral illnesses are a result of exposure, cellular infection andreplication of the virus to viral levels that overpower the host'simmune system leading to signs and symptoms of disease. Viruses can'tmake new viruses on their own and require host cells to produce newviruses. With low levels of viral exposure, this typically results inthe body's immune system to activate and attack the virus directly priorto symptom development. This results in the virus being eliminated fromthe host. In people with limited immune response and/or high levels ofviral exposure, the host immune system will most likely becomeoverpowered by the viral load and the patient will become ill. Viralinfection propagates as the virus attaches to the cell surface at whichtime it is brought into the cell. Viruses bring in their own DNA or RNAinstructions into the cell for replication and their eventual releaseonly to repeat the process in greater numbers again and again increasingthe infection rate, which increases the viral load and eventuallyleading to severe illness.

Viruses are known to have several cellular binding proteins called“spike proteins” on the surface which seek out and attach to the cellsurface. For the SARS-CoV and SARS-CoV-2 viruses, this attachment can beto the ACE2 receptor and other accessory receptors like GRP78, which arefound on the cell surface of respiratory lung and endothelial cells.These receptors then transport the viral material into the cells whereit is replicated and expelled from the infected cells to repeat theprocess.

Method to Select and Inhibit Viruses with a GRP78 Recognition Site.

Surface bound GRP78 has been reported to be important for attachment andentry into host cells for several different types of viruses. InElfiky's publication, they predict that coronaviruses SARS-CoV-2, NL63,229E, OC43, and HKU1 have a similar GRP78 recognition/binding site.Other publications have shown that surface bound GRP78 is important forattachment and entry of Zika virus, Ebola virus, MERS-CoV,Coxsackievirus A9, Dengue virus, Japanese encephalitis virus (JEV) andInfluenza viruses but have not examined if they have the same GRP78binding sequences. From our previous publications, we demonstrated thatthe sequence from kringle 5 of human plasminogen, CYTTNPRKLYDYC bindstightly to surface bound GRP78.

Although, Elfiky's prediction uses a weaker GRP78 binding sequence,PEP42 (CTVALPGGYVRVC), than the K5 sequence, it still retains some ofthe important amino acids that we had shown previously for GRP78 bindingin the kringle 5 structural fold region. In FIG. 17, we show sequencealignment of viruses compared to our known GRP78 binding kringlesequences to identify possible GRP78 binding sites. Using thisalignment, we can predict which viruses will be responsive to our methodof treatment for viral inhibition through blocking the N-terminal domainof GRP78. From this alignment, we can now predict that SARS-CoV-2, NL63,229E, OC43, HKU1, MERS-CoV, EBOLA, Zika, Yellow Fever, West Nile, andInfluenza A, B viruses will be inhibited by our therapies due to theirGRP78 binding. However, SARS-CoV does not contain the proposed GRP78binding sequence so we predict that our GRP78 inhibitors would not beeffective against this virus. As proof this prediction is accurate, inExample 6, we can show that our N-terminal binding GRP78 inhibitor,Kr1Fc, potently blocks attachment and entry of SARS-CoV-2 pseudoviruses,but we have recently discovered that our GRP78 inhibitor, Kr1Fc, did notblock viral induced death or weight loss in mice infected with a lethalvariant of SARS-CoV.

Method for Selecting Our Target Population for Prophylactic orTherapeutic Treatment with GRP78 Inhibitors Against SARS-CoV-2 and OtherViruses Infection

People under the age of 65 with comorbidities like cancer, obesity,cardiac disease, lung disease, and hypertension, have a higher risk ofbeing infected with SARS-CoV-2 virus than those without a comorbidity.This same group of people also has a much higher rate of mortality fromCOVID19. For people over 65 with or without a comorbidity their chancesof dying from COVID19 are also much higher than those under the age of65. Due to stress applied to endothelial, lung, respiratory and immunecells by these comorbidities and in general the aging process, thepresence of surface bound GRP78 has recently been recognized as animportant player in aging and disease progression.

With aging and with comorbidities, cells become more stressed leading toconsistent and higher expression of cell surface bound GRP78. Thishigher expression of the GRP78 allows for more viral binding and entryinto cells (FIG. 1). Viral replication and release of the viralparticles can then occur at a much higher rate. This process ofincreased binding, entry and replication of viruses due to increasedGRP78 receptor can then overpower the immune system leading to increasedmorbidity and mortality.

Treating Patients with Autoimmune Diseases

Patients with autoimmune diseases do not have a fully functioning immunesystem and have been reported to present with higher expression ofsurface bound GRP78. Current treatments and vaccines that are designedto stimulate the immune system against viruses like SARS-CoV-2 are muchless effective in these patients that are prone to unwanted increasedviral progression. Providing prophylactic and/or therapeutic supportwith our GRP78 inhibitors to these patients may provide a level ofprotection against infection and if infected will block new viralattachment, entry, replication and release of new viral particles intothe patient.

Treating Patients with Cancer

Patients with cancer, especially lung and blood borne cancers, have beenshown to have a higher expression of surface bound GRP78 on their lowerand higher respiratory tract cells as well as their endothelial cells.These patients, because of their chemotherapy treatments, usually have areduced immune response to vaccines and pathogens. By treating thesepatients, either prophylactically or after infection SARS-CoV-2 virus,with our GRP78 inhibitions may allow for a decreased chance forinfection and a decreased rate of viral infectivity by blocking virusaccess to the surface bound GRP78 receptor.

Treating Patients with Obesity.

Patients with obesity have several comorbidities like hypertension,diabetes, and poor circulation which can cause stressed lung andendothelial cells resulting in increased surface expressed GRP78. Thisincrease in surface expressed GRP78 makes this group of people at highrisk for SARS-CoV-2 infection and death. Using our GRP78 inhibitors toblock the amount of viral infectivity through surface bound GRP78, wemay greatly reduce symptoms and the severity of the viral infection.

Treating Patients with Diabetes

Patients with diabetic type 1 or type 2 disease have been shown to havehigher expression of surface bound GRP78 on kidney and endothelialcells. With the use of our GRP78 inhibitors in these patients, the viralload and chance of SARS-CoV-2 infection should be greatly reducedallowing for normal immune attack and elimination of viral particles.

Treating Patients with Cardiovascular Disease

A hallmark for patients with cardiovascular disease is increasedendoplasmic stress in heart tissues leading to higher expression of ACE2and surface bound GRP78 on their vasculature cells. Although, increasedACE2 expression allows for increased SARS-CoV-2 attachment, thisinvention teaches that SARS-CoV-2 requires surface bound GRP78 for viralentry and as such, by reducing the surface expression of GRP78 with ourGRP78 inhibitors, this should lead to reduced viral infection andmortality.

Treating Patients with Hypertension

Studies show that patients with hypertension have increased endoplasmicreticulum expressed GRP78 in their arterial cells. This increase inGRP78 expression leads to an increase in surface bound GRP78. As listedabove, surface expression of GRP78 allows for higher SARS-CoV-2 viralattachment, entry and replication in cells. By reducing the expressionof surface bound GRP78 with our inhibitors, not only will they reducethe risk for viral infection but also reduce chronic hypertensioneffects.

Treating Patients Over 65 Years of Age

With aging, inflammation is more prevalent, which leads to muscle andarterial cells being more stressed resulting in consistent expression ofsurface bound GRP78. It is clear that SARS-CoV-2 infection severityincreases substantially with age. This higher level of GRP78 receptorallows for availability of more attachment and entry of SARS-CoV-2 virusinto the cells The treatment of patients over the age of 65 with GRP78inhibitors will lead to reduced surface bound GRP78 on stressedunhealthy cells. With less receptors for SARS-CoV-2 virus attachment,the less severe the SARS-CoV-2 infection will be.

It is rare that young (<60), heathy people show severe symptoms fromSARS-CoV-2 infection. We believe this is due to the fact that surfacebound GRP78 on young, heathy people's cells is very low, which decreasesthe risk of viral attachment. This limits the severity of infection andgreatly decreases the mortality rate of children and younger people. Assuch, there is no need to treat this population with our GRP78inhibitors.

However, there are exceptions to this theory. For example, Influenzaviruses infect the young and the old even though the viruses contain aGRP78 binding site and there is no surface bound GRP78 on their cells.Recent publications show that infected cells induce surface GRP78expression, which leads to more infection. In these cases, treatmentwith GRP78 inhibitors will reduce surface bound GRP78, resulting indecreased viral load and a reduced time of infectivity.

Types of Treatment

For our GRP78 inhibitors, this invention shows two type of treatmentoptions. The first is to give our GRP78 inhibitors as a treatment forpatients listed above after SARS-CoV-2 infection. Infected patients withcomorbidities will benefit from decreased surface bound GRP78 resultingin decreased virus attachment and entry.

The second method to use for our GRP78 inhibitors is to give themprophylactically. For the patients listed above with comorbidities andfor those above 65 years of age whose lung, respiratory and arterialcells have increased surface bound GRP78 expression, treatment with ourGRP78 inhibitors prophylactically will reduce the risk of SARS-CoV-2viral infection and disease progression. Our GRP78 inhibitors givenprophylactically will may also be effective at protecting health careworkers, first responders, people who travel to high infection areas andteachers from SARS-CoV-2 infection.

Co-Treatment Options

This invention shows that our GRP78 inhibitors may be given incombination with other types of therapies to help reduce viral infectionand the resulting symptoms. For example:

-   -   anti-inflammatory agents like Dexamethasone have been shown to        reduce the symptoms like cytokine inflammation leading to        increased survival,    -   anti-viral agents like Remdesivir which has been shown to block        replication of viruses leading to reduced viral disease.    -   immune suppressive agents like Baricitinib (JAK inhibitor), and        Tocilzumab (IL6 inhibitor) which have not been shown to be        effective in late-stage disease but show promise in early to        mid-stage disease.    -   anti-coagulant agents like heparin which significantly reduced        the symptoms of clot storms as a result of SARS-CoV-2 infection.    -   vaccines like those against SARS-CoV-2, or influenza may be        effective against the main strain but not against evolving        variants. By using both vaccines and GRP78 inhibitors, they may        be more effective for cures.

Problems with Current Therapies and Vaccines Against SARS-CoV-2 Virus:

Vaccines against the SARS-CoV-2 virus are designed to force the immunesystem to attack the virus through either the spike protein on the virussurface or other envelope proteins. This then allows the body to makeantibodies against the SARS-CoV-2 virus spike protein, which blockSARS-CoV-2 virus attaching and internalizing into the host cell.Currently, there are three approved vaccines against the spike proteinof SARS-CoV-2 virus. For people with a weaken immune system due tocomorbidities or other drug treatments, these vaccines may not aseffective. Also SARS-CoV-2 variant (B.1.351) infections have been shownto be resistant to the AstraZeneca Covid-19 vaccine for mild-to-moderateinfections. This is the underlying issue with anti-viral vaccines inthat there is a need for yearly updated versions of the vaccine tocombat evolving variants.

Monoclonal antibodies against SARS-CoV-2 spike protein have beenapproved for emergency use by the FDA. These antibodies have been shownto be effective against patients with early SARS-CoV-2 infections.Patients that were hospitalized did not show any improvement. Finally,like vaccines these therapies are very targeted against the viral spikeprotein and variants may not be inhibited.

Remdesivir is a protease inhibitor that has been approved for use by theFDA. It also failed against hospitalized patients and did not preventdeath. If give early in the SARS-CoV-2 viral infection, it was shown todecrease hospitalization time and duration of the infection.

Listed above are the only FDA approved therapies for COVID19 disease asof the writing of this invention. However, several others areprogressing through clinical studies that show some promise. Baricitinibis a Janus Kinase inhibitor that is an immunosuppressant. This therapyhas shown to be effective against SARS-CoV-2 virus infected patients onoxygen but not on ventilation. Again, late-stage COVID19 disease is notaddressed. Tocilizumab is a monoclonal antibody against IL-6. InhibitingIL-6 treats SARS-CoV-2 infection symptoms by tamping down inflammationallowing the immune system to function better. The outcomes fromlate-stage patients receiving Tocilzumab were reduced time to dischargefor both people on ventilation and oxygen.

This invention teaches a method for treating patients with SARS-CoV-2infection that uses N-terminal GRP78 inhibitors. Unlike the vaccines orcurrent viral treatments, our therapy addresses the potential viralinfection at the cell surface, not at the virus surface. By inhibitingthe host cells receptors and not the virus, mutant variants will not beresistant to GRP78 inhibitor therapy. Also, since GRP78 inhibitors donot rely on the immune system for viral clearance or inhibition, immunesuppressed patients can still be treated. As such, late stage COVID19diseased patients, patients with suppressed immune systems or patientswith comorbidities will still be sensitive to our GRP78 inhibitors,which will reduce infection time leading to less hospital stay and viralload.

As shown in FIG. 17, FIG. 17 is a table showing Protein sequencecomparisons for GRP78 binding site a comparison shows that selectedprotein sequences (indicated in a red color in FIG. 17) are identical toKringle 5 sequence and a second category (yellow) is a similar type ofamino acid. Where there is neither red nor yellow, it is concluded thereis no similarity. All kringle and virus sequence comparisons arecompared to Kringle 5. An asterisk (*) in FIG. 17 marks the amino acidsimportant for GRP78 binding, in columns 3, 8, 12 and 18. Proteinsequence comparisons for GRP78 binding site. C is identical to Kringle 5sequence, yellow is similar type of amino acid, white is no similarity.All kringle and virus sequence comparisons are compared to Kringle 5. *marks the amino acids important for GRP78 binding.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof such claims as shall be appended.

In accordance with my invention, I claim:
 1. A method for the treatmentcomprising administering to a person in need an effective dose of theantivirus agent of claim 1 for prevention (prophylactic) and inhibition(treatment) of viral infection for viruses that use surface-bound GRP78for attachment, entry and replication like SARS-CoV-2 virus, SARS-CoVvirus, MER-CoV virus, Japanese Encephalitis Virus, Coxsackievirus,Dengue virus and Influenza A&B viruses.
 2. The antivirus agent of claim1, comprising: a GRP78 antagonist or a pharmaceutically acceptable salt:wherein the GRP78 antagonist is selected from a group consisting of aplasminogen kringle 5 fragment, a plasminogen kringle 5 fragmentattached to immunoglobulin, an ROR1 kringle fragment, an ROR1 kringlefragment attached to an immunoglobulin, an ROR2 kringle fragment, and anROR2 kringle fragment attached to an immunoglobulin.
 3. The antivirusagent of claim 2, wherein the plasminogen kringle 5 fragment is of SEQID NO:1 and combinations thereof.
 4. The antivirus agent of claim 2,wherein the plasminogen kringle 5 fragment attached to immunoglobulin isselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and combinationsthereof.
 5. The antivirus agent of claim 2, wherein the ROR1 kringlefragment is of SEQ ID NO:19, and combinations thereof.
 6. The antivirusagent of claim 2, wherein the ROR1 kringle fragment attached toimmunoglobulin is selected from the group consisting of SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, and combinations thereof.
 7. The antivirusagent of claim 2, wherein the ROR2 kringle fragment is SEQ ID NO:33 andcombinations thereof.
 8. The antivirus agent of claim 2, wherein theROR2 kringle fragment attached to immunoglobulin is selected from thegroup consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and combinationsthereof.
 9. The antivirus agent of claim 2, wherein the GRP78 antagonistbinds to the N-terminal GRP78 domain and competes with claim 1 GRP78antagonists to induce GRP78 internalization.
 10. An antivirus agentcomprising: a GRP78 antagonist or a pharmaceutically acceptable salt:wherein the GRP78 antagonist is selected from a group consisting of aplasminogen kringle 5 fragment, a plasminogen kringle 5 fragmentattached to immunoglobulin, an ROR1 kringle fragment, an ROR1 kringlefragment attached to an immunoglobulin, an ROR2 kringle fragment, and anROR2 kringle fragment attached to an immunoglobulin.
 11. The antivirusagent of claim 10, wherein the plasminogen kringle 5 fragment is of SEQID NO:1 and combinations thereof.
 12. The antivirus agent of claim 10,wherein the plasminogen kringle 5 fragment attached to immunoglobulin isselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, and combinationsthereof.
 13. The antivirus agent of claim 10, wherein the ROR1 kringlefragment is of SEQ ID NO:19, and combinations thereof.
 14. The antivirusagent of claim 10, wherein the ROR1 kringle fragment attached toimmunoglobulin is selected from the group consisting of SEQ ID NO:20,SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30,SEQ ID NO:31, SEQ ID NO:32, and combinations thereof.
 15. The antivirusagent of claim 10, wherein the ROR2 kringle fragment is SEQ ID NO:33 andcombinations thereof.
 16. The antivirus agent of claim 10, wherein theROR2 kringle fragment attached to immunoglobulin is selected from thegroup consisting of SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ IDNO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and combinationsthereof.
 17. The antivirus agent of claim 10, wherein the GRP78antagonist binds to the N-terminal GRP78 domain and competes with claim1 GRP78 antagonists to induce GRP78 internalization.